All about Drugs, live, by DR ANTHONY MELVIN CRASTO, Worlddrugtracker, OPEN SUPERSTAR Helping millions, 10 million hits on google, pushing boundaries,2.5 lakh plus connections worldwide, 24 lakh plus VIEWS on this blog in 221 countries, 7 CONTINENTS The views expressed are my personal and in no-way suggest the views of the professional body or the company that I represent, USE CTRL AND+ KEY TO ENLARGE BLOG VIEW……………………A 90 % paralysed man in action for you, I am suffering from transverse mylitis and bound to a wheel chair, With death on the horizon, I have lot to acheive

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DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 30 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international,
etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules
and implementation them on commercial scale over a 30 year tenure till date Dec 2017, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 50 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 19 lakh plus views on New Drug Approvals Blog in 216 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

In August 2019, Suzhou Neupharma and its licensee Checkpoint Therapeutics are developing CK-101 (phase II clinical trial), a novel third-generation, covalent, EGFR inhibitor, as a capsule formulation, for the treatment of cancers including NSCLC and other advanced solid tumors. In September 2017, the FDA granted Orphan Drug designation to this compound, for the treatment of EGFR mutation-positive NSCLC; in January 2018, the capsule was being developed as a class 1 chemical drug in China.

CK-101 (RX-518), a small-molecule inhibitor of epidermal growth factor receptor (EGFR), is in early clinical development at Checkpoint Therapeutics and Suzhou NeuPharma for the potential treatment of EGFR-mutated non-small cell lung cancer (NSCLC) and other advanced solid malignancies.

In 2017, the product was granted orphan drug designation in the U.S. for the treatment of EGFR mutation-positive NSCLC.

There are at least 400 enzymes identified as protein kinases. These enzymes catalyze the phosphorylation of target protein substrates. The phosphorylation is usually a transfer reaction of a phosphate group from ATP to the protein substrate. The specific structure in the target substrate to which the phosphate is transferred is a tyrosine, serine or threonine residue. Since these amino acid residues are the target structures for the phosphoryl transfer, these protein kinase enzymes are commonly referred to as tyrosine kinases or serine/threonine kinases.

[0003] The phosphorylation reactions, and counteracting phosphatase reactions, at the tyrosine, serine and threonine residues are involved in countless cellular processes that underlie responses to diverse intracellular signals (typically mediated through cellular receptors), regulation of cellular functions, and activation or deactivation of cellular processes. A cascade of protein kinases often participate in intracellular signal transduction and are necessary for the realization of these cellular processes. Because of their ubiquity in these processes, the protein kinases can be found as an integral part of the plasma membrane or as cytoplasmic enzymes or localized in the nucleus, often as components of enzyme complexes. In many instances, these protein kinases are an essential element of enzyme and structural protein complexes that determine where and when a cellular process occurs within a cell.

[0004] The identification of effective small compounds which specifically inhibit signal transduction and cellular proliferation by modulating the activity of tyrosine and serine/threonine kinases to regulate and modulate abnormal or inappropriate cell proliferation, differentiation, or metabolism is therefore desirable. In particular, the identification of compounds that specifically inhibit the function of a kinase which is essential for processes leading to cancer would be beneficial.

[0005] While such compounds are often initially evaluated for their activity when dissolved in solution, solid state characteristics such as polymorphism are also important. Polymorphic forms of a drug substance, such as a kinase inhibitor, can have different physical properties, including melting point, apparent solubility, dissolution rate, optical and mechanical properties, vapor pressure, and density. These properties can have a direct effect on the ability to process or manufacture a drug substance and the drug product. Moreover, differences in these properties

can and often lead to different pharmacokinetics profiles for different polymorphic forms of a drug. Therefore, polymorphism is often an important factor under regulatory review of the ‘sameness’ of drug products from various manufacturers. For example, polymorphism has been evaluated in many multi-million dollar and even multi-billion dollar drugs, such as warfarin sodium, famotidine, and ranitidine. Polymorphism can affect the quality, safety, and/or efficacy of a drug product, such as a kinase inhibitor. Thus, there still remains a need for polymorphs of kinase inhibitors. The present disclosure addresses this need and provides related advantages as well.

[0258] Crude compound of Formula I (~30 g, 75% of weight based assay) was dissolved in ethyl acetate (3 L) at 55-65 °C under nitrogen. The resulting solution was filtered via silica gel pad and washed with ethyl acetate (3 L><2) at 55-65 °C. The filtrate was concentrated via vacuum at 30-40 °C to ~2.4 L. The mixture was heated up to 75-85 °C and maintained about 1 hour.

Then cooled down to 50-60 °C and maintained about 2 hours. The heat-cooling operation was repeated again and the mixture was then cooled down to 20-30 °C and stirred for 3 hours. The resulting mixture was filtered and washed with ethyl acetate (60 mL><2). The wet cake was dried via vacuum at 30-40 °C to get (about 16 g) of the purified Form I of the compound of Formula I.

Example 3. Preparation of Form III of the compound of Formula I

[0259] The compound of Formula I (2 g) was dissolved in EtOH (40 mL) at 75-85 °C under nitrogen. n-Heptane (40 mL) was added dropwise into reaction at 75-85 °C. The mixture was stirred at 75-85 °C for 1 hour. Then cooled down to 50-60 °C and maintained about 2 hours. The heat-cooling operation was repeated again and continued to cool the mixture down to 20-30 °C and stirred for 3 hours. The resulting mixture was filtered and washed with EtOH/n-Heptane (1/1, 5 mL><2). The wet cake was dried via vacuum at 30-40 °C to get the purified Form III of the compound of Formula I (1.7 g).

Example 4. Preparation of Form IV of the compound of Formula I The crude compound of Formula I (15 g) was dissolved in ethyl acetate (600 mL) at 75-85 °C under nitrogen and treated with anhydrous Na2S04, activated carbon, silica metal scavenger for 1 hour. The resulting mixture was filtered via neutral Al203 and washed with ethyl acetate (300 mL><2) at 75-85 °C. The filtrate was concentrated under vacuum at 30-40 °C and swapped with DCM (150 mL). n-Heptane (75 mL) was added into this DCM solution at 35-45 °C, and then the mixture was cooled down to 20-30 °C slowly. The resulting mixture was filtered and washed with DCM/n-Heptane (2/1, 10 mL><3). The wet cake was dried via vacuum at 35-40 °C to get the purified Form IV of the compound of Formula I (9.6 g).

Example 5. Preparation of Form V of the compound of Formula I

[0260] Polymorph Form III of the compound of Formula I was dried in oven at 80 °C for 2 days to obtain the polymorph Form V.

Example 6. Preparation of Form VI of the compound of Formula I

[0261] The compound of Formula I (1 g) was dissolved in IPA (20 mL) at 75-85 °C under nitrogen. n-Heptane (20 mL) was added dropwise into reaction at 75-85 °C. The mixture was stirred at 45-55 °C for 16 hours. Then heated up to 75-85 °C and maintained about 0.5 hour.

Then cooled down to 45-55 °C for 0.5 hour and continued to cool the mixture down to 20-30 °C and stirred for 3 hours. Filtered and washed with IPA/n-Heptane (1/1, 3 mL><2). The wet cake was dried via vacuum at 75-80 °C for 2 hours to get the purified Form VI of the compound of Formula I.

Example 7. Preparation of Form VIII of the compound of Formula I

[0262] The polymorph Form VI of the compound of Formula I was dried in oven at 80 °C for 2 days to obtain the polymorph Form VIII.

Example 8. X-ray powder diffraction (XRD)

[0263] X-ray powder diffraction (XRD) patterns were obtained on a Bruker D8 Advance. A CuK source (=1.54056 angstrom) operating minimally at 40 kV and 40 mA scans each sample between 4 and 40 degrees 2-theta. The step size is 0.05°C and scan speed is 0.5 second per step.

Example 9. Thermogravimetric Analyses (TGA)

[0264] Thermogravimetric analyses were carried out on a TA Instrument TGA unit (Model TGA 500). Samples were heated in platinum pans from ambient to 300 °C at 10 °C/min with a nitrogen purge of 60mL/min (sample purge) and 40mL/min (balance purge). The TGA temperature was calibrated with nickel standard, MP=354.4 °C. The weight calibration was performed with manufacturer-supplied standards and verified against sodium citrate dihydrate desolvation.

Example 10. Differential scanning calorimetry (DSC)

[0265] Differential scanning calorimetry analyses were carried out on a TA Instrument DSC unit (Model DSC 1000 or 2000). Samples were heated in non-hermetic aluminum pans from ambient to 300 °C at 10 °C/min with a nitrogen purge of 50mL/min. The DSC temperature was calibrated with indium standard, onset of l56-l58°C, enthalpy of 25-29J/g.

Example 11. Hygroscopicity (DVS)

[0266] The moisture sorption profile was generated at 25°C using a DVS Moisture Balance Flow System (Model Advantage) with the following conditions: sample size approximately 5 to 10 mg, drying 25°C for 60 minutes, adsorption range 0% to 95% RH, desorption range 95% to 0% RH, and step interval 5%. The equilibrium criterion was <0.01% weight change in 5 minutes for a maximum of 120 minutes.

Example 12: Microscopy

[0267] Microscopy was performed using a Leica DMLP polarized light microscope equipped with 2.5X, 10X and 20X objectives and a digital camera to capture images showing particle shape, size, and crystallinity. Crossed polars were used to show birefringence and crystal habit for the samples dispersed in immersion oil.

Example 13: HPLC

[0256] HPLCs were preformed using the following instrument and/or conditions.

Eli Lilly is developing SY 008, a sodium glucose transporter 1 (SGLT1) inhibitor, for the treatment of diabetes mellitus. The approach of inhibiting SGLT1 could be promising because it acts independently of the beta cell and could be effective in both early and advanced stages of diabetes. Reducing both glucose and insulin may improve the metabolic state and potentially the health of beta cells, without causing weight gain or hypoglycaemia. Clinical development is underway in Singapore and China.

As at August 2018, no recent reports of development had been identified for phase-I development in Diabetes-mellitus in Singapore (PO).

Suzhou Yabao , under license from Eli Lilly , is developing SY-008 , an SGLT1 inhibitor, for the potential oral capsule treatment of type 2 diabetes in China. By April 2019, a phase Ia trial was completed

The present invention is in the field of treatment of diabetes and other diseases and disorders associated with hyperglycemia. Diabetes is a group of diseases that is characterized by high levels of blood glucose. It affects approximately 25 million people in the United States and is also the 7th leading cause of death in U.S. according to the 201 1 National Diabetes Fact Sheet (U.S. Department of Health and Human Services, Centers for Disease Control and Prevention). Sodium-coupled glucose cotransporters (SGLT’s) are one of the transporters known to be responsible for the absorption of carbohydrates, such as glucose. More specifically, SGLTl is responsible for transport of glucose across the brush border membrane of the small intestine. Inhibition of SGLTl may result in reduced absorption of glucose in the small intestine, thus providing a useful approach to treating diabetes.

U.S. Patent No. 7,655,632 discloses certain pyrazole derivatives with human SGLTl inhibitory activity which are further disclosed as useful for the prevention or treatment of a disease associated with hyperglycemia, such as diabetes. In addition, WO 201 1/039338 discloses certain pyrazole derivatives with SGLT1/SGLT2 inhibitor activity which are further disclosed as being useful for treatment of bone diseases, such as osteoporosis.

There is a need for alternative drugs and treatment for diabetes. The present invention provides certain novel inhibitors of SGLTl which may be suitable for the treatment of diabetes.

Borane-dimethyl sulfide complex (2M in THF; 1 16 mL, 0.232 mol) is added slowly to a solution of 4-bromo-2-methylbenzoic acid (24.3 g, 0.1 13 mol) in anhydrous tetrahydrofuran (THF, 146 mL) at 3 °C. After stirring cold for 10 min the cooling bath is removed and the reaction is allowed to warm slowly to ambient temperature. After 1 hour, the solution is cooled to 5°C, and water (100 mL) is added slowly. Ethyl acetate (100 mL) is added and the phases are separated. The organic layer is washed with saturated aqueous NaHC03 solution (200 mL) and dried over Na2S04. Filtration and concentration under reduced pressure gives a residue which is purified by filtration through a short pad of silica eluting with 15% ethyl acetate/iso-hexane to give the title compound (20.7 g, 91.2% yield). MS (m/z): 183/185 (M+l-18).

A solution of 4-bromo- 1 -chloromethyl-2-methyl-benzene (13.16 g, 59.95 mmoles) in acetonitrile (65.8 mL) is prepared. Potassium carbonate (24.86 g, 179.9 mmol), potassium iodide (1 1.94 g, 71.94 mmol) and methyl 4-methyl-3-oxo valerate (8.96 mL; 62.95 mmol) are added. The resulting mixture is stirred at ambient temperature for 20 hours. Hydrochloric acid (2N) is added to give pH 3. The solution is extracted with ethyl acetate (100 ml), the organic phase is washed with brine (100 ml) and dried over Na2S04. The mixture is filtered and concentrated under reduced pressure. The residue is dissolved in toluene (65.8 mL) and hydrazine monohydrate (13.7 mL, 0.180 mol) is added. The resulting mixture is heated to reflux and water is removed using a Dean and Stark apparatus. After 3 hours the mixture is cooled to 90 °C and additional hydrazine monohydrate (13.7 mL; 0.180 mol) is added and the mixture is heated to reflux for 1 hour. The mixture is cooled and concentrated under reduced pressure. The resulting solid is triturated with water (200 mL), filtered and dried in a vacuum oven over P2O5 at 60°C. The solid is triturated in iso-hexane (200 mL) and filtered to give the title compound (14.3 g; 77.1% yield). MS (m/z): 309/31 1 (M+l).

(32.18 g, 232.9 mmol), dichloromethane (250 mL) and water (120 mL) are combined and the mixture is stirred at ambient temperature for 18 hours. The mixture is partitioned between dichloromethane (250 mL) and water (250 mL). The organic phase is washed with brine (250 mL), dried over Na2S04, filtered, and concentrated under reduced pressure. The resulting residue is purified by flash chromatography (eluting with 10% ethyl acetate in dichloromethane to 70% ethyl acetate in dichloromethane) to give the title compound (36.5 g, 74% yield). MS (m/z): 639/641 (M+l).

The title compound is prepared essentially by first treating the compound of Prearation 14 with HC1 as discussed in Preparation 15 then treating the resulting hydrochloride salt with triethyl amine as discussed in the first alternative synthesis of Example 1. MS (m/z): 598.8, 599.8 (M+l), 596.8, 597.8 (M-l).

Scheme 3, step D: Trifluoroacetic acid (32.2 mL; 0.426 mol) is added to a solution of tert-butyl 2- {(3JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-beta-D-glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (14.87 g; 21.28 mmol) in dichloromethane (149 mL) cooled in iced water. The solution is allowed to warm to room temperature. After 30 minutes, the mixture is slowly added to ammonia in MeOH (2M; 300 mL), applying cooling as necessary to maintain a constant temperature. The solution is stirred at room temperature for 15 min. The mixture is concentrated under reduced pressure and the residue is purified using SCX-2 resin. The basic filtrate is concentrated under reduced pressure and the residue is triturated/sonicated in ethyl acetate, filtered and dried. The resulting solid is dissolved in MeOH (200ml) and concentrated in vacuo. This is repeated several times give the title compound (12.22 g, yield 96%). MS (m/z): 599 (M+l). [a]D20 = -12 ° (C=0.2, MeOH).

The present invention relates to a novel SGLT1 inhibitor which is an acetate salt of a pyrazole compound, to pharmaceutical compositions comprising the compound, to methods of using the compound to treat physiological disorders, and to intermediates and processes useful in the synthesis of the compound.

The present invention is in the field of treatment of diabetes and other diseases and disorders associated with hyperglycemia. Diabetes is a group of diseases that is characterized by high levels of blood glucose. It affects approximately 25 million people in the United States and is also the 7th leading cause of death in U.S. according to the 2011 National Diabetes Fact Sheet (U.S. Department of Health and Human Services, Centers for Disease Control and Prevention). Sodium-coupled glucose cotransporters (SGLT’s) are one of the transporters known to be responsible for the absorption of carbohydrates, such as glucose. More specifically, SGLT1 is responsible for transport of glucose across the brush border membrane of the small intestine. Inhibition of SGLT1 may result in reduced absorption of glucose in the small intestine, thus providing a useful approach to treating diabetes.

U.S. Patent No. 7,655,632 discloses certain pyrazole derivatives with human SGLT1 inhibitory activity which are further disclosed as useful for the prevention or treatment of a disease associated with hyperglycemia, such as diabetes. In addition, WO 2011/039338 discloses certain pyrazole derivatives with SGLT1/SGLT2 inhibitor activity which are further disclosed as being useful for treatment of bone diseases, such as osteoporosis.

There is a need for alternative drugs and treatment for diabetes. The present invention provides an acetate salt of a pyrazole compound, which is an SGLT1 inhibitor, and as such, may be suitable for the treatment of certain disorders, such as diabetes. Accordingly, the present invention provides a compound of Formula I:

Borane-dimethyl sulfide complex (2M in THF; 116 mL, 0.232 mol) is added slowly to a solution of 4-bromo-2-methylbenzoic acid (24.3 g, 0.113 mol) in anhydrous tetrahydrofuran (THF, 146 mL) at 3 °C. After stirring cold for 10 min the cooling bath is removed and the reaction is allowed to warm slowly to ambient temperature. After 1 hour, the solution is cooled to 5°C, and water (100 mL) is added slowly. Ethyl acetate (100 mL) is added and the phases are separated. The organic layer is washed with saturated aqueous NaHC03 solution (200 mL) and dried over Na2S04. Filtration and concentration under reduced pressure gives a residue which is purified by filtration through a short pad of silica eluting with 15% ethyl acetate/iso-hexane to give the title compound (20.7 g, 91.2% yield). MS (m/z): 183/185 (M+l-18).

A solution of 4-bromo-l-chloromethyl-2-methyl-benzene (13.16 g, 59.95 mmoles) in acetonitrile (65.8 mL) is prepared. Potassium carbonate (24.86 g, 179.9 mmol), potassium iodide (11.94 g, 71.94 mmol) and methyl 4-methyl-3-oxovalerate (8.96 mL; 62.95 mmol) are added. The resulting mixture is stirred at ambient temperature for 20 hours. Hydrochloric acid (2N) is added to give pH 3. The solution is extracted with ethyl acetate (100 ml), the organic phase is washed with brine (100 ml) and dried over Na2S04. The mixture is filtered and concentrated under reduced pressure. The residue is dissolved in toluene (65.8 mL) and hydrazine monohydrate (13.7 mL, 0.180 mol) is added. The resulting mixture is heated to reflux and water is removed using a Dean and Stark apparatus. After 3 hours the mixture is cooled to 90 °C and additional hydrazine monohydrate (13.7 mL; 0.180 mol) is added and the mixture is heated to reflux for 1 hour. The mixture is cooled and concentrated under reduced pressure. The resulting solid is triturated with water (200 mL), filtered and dried in a vacuum oven over P2Os at 60°C. The solid is triturated in iso-hexane (200 mL) and filtered to give the title compound (14.3 g; 77.1% yield). MS (m/z): 309/311 (M+l).

Scheme 3, step D: Trifluoroacetic acid (32.2 mL; 0.426 mol) is added to a solution of tert-butyl 2-{(3JE)-4-[3-methyl-4-({5-(propan-2-yl)-3-beta-D- glucopyranosyl)oxy]-lH-pyrazol-4-yl}methyl)phenyl]but-3-en-l-yl}-2,9- diazaspiro[5.5]undecane-9-carboxylate (14.87 g; 21.28 mmol) in dichloromethane (149 mL) cooled in iced water. The solution is allowed to warm to room temperature. After 30 minutes, the mixture is slowly added to ammonia in MeOH (2M; 300 mL), applying cooling as necessary to maintain a constant temperature. The solution is stirred at room temperature for 15 min. The mixture is concentrated under reduced pressure and the residue is purified using SCX-2 resin. The basic filtrate is concentrated under reduced pressure and the residue is triturated/sonicated in ethyl acetate, filtered and dried. The resulting solid is dissolved in MeOH (200mL) and concentrated in vacuo. This is repeated several times to give the title compound (free base) (12.22 g, yield 96%). MS (m/z): 599 (M+l); [a]D20 = -12 ° (C=0.2, MeOH).

4- {4- [(1 E)-4-(2,9-diazaspiro [5.5]undec-2-yl)but- 1 -en- 1 -yl] -2-methylbenzyl } -5 – (propan-2-yl)-lH-pyrazol-3-yl beta-D-glucopyranoside (902 mg) is placed in a round bottom flask (100 mL) and treated with wet ethyl acetate (18 mL). [Note – wet ethyl acetate is prepared by mixing ethyl acetate (100 mL) and dionized water (100 mL). After mixing, the layers are allowed to separate, and the top wet ethyl acetate layer is removed for use. Acetic acid is a hydrolysis product of ethyl acetate and is present in wet ethyl acetate.] The compound dissolves, although not completely as wet ethyl acetate is added. After several minutes, a white precipitate forms. An additional amount of wet ethyl acetate (2 mL) is added to dissolve remaining compound. The solution is allowed to stir uncovered overnight at room temperature during which time the solvent partially evaporates. The remaining solvent from the product slurry is removed under vacuum, and the resulting solid is dried under a stream of nitrogen to provide the final title compound as a crystalline solid. A small amount of amorphous material is identified in the product by solid-state NMR. This crystalline final title compound may be used as seed crystals to prepare additional crystalline final title compound.

The present invention is in the field of treatment of diabetes and other diseases and conditions associated with hyperglycemia. Diabetes is a group of diseases characterized by high blood sugar levels. It affects approximately 25 million people in the United States, and according to the 2011 National Diabetes Bulletin, it is also the seventh leading cause of death in the United States (US Department of Health and Human Resources Services, Centers for Disease Control and Prevention). Sodium-coupled glucose cotransporters (SGLT’s) are one of the transporters known to be responsible for the uptake of carbohydrates such as glucose. More specifically, SGLT1 is responsible for transporting glucose across the brush border membrane of the small intestine. Inhibition of SGLT1 can result in a decrease in glucose absorption in the small intestine, thus providing a useful method of treating diabetes.

Alternative medicines and treatments for diabetes are needed. The present invention provides an acetate salt of a pyrazole compound which is an SGLT1 inhibitor, and thus it is suitable for treating certain conditions such as diabetes.

U.S. Patent No. 7,655,632 discloses certain pyrazole derivatives having human SGLT1 inhibitory activity, which are also disclosed for use in the prevention or treatment of diseases associated with hyperglycemia, such as diabetes. Moreover, WO 2011/039338 discloses certain pyrazole derivatives having SGLT1/SGLT2 inhibitor activity, which are also disclosed for use in the treatment of bone diseases such as osteoporosis.

Diabetes is a group of lifelong metabolic diseases characterized by multiple causes of chronic hyperglycemia. Long-term increase in blood glucose can cause damage to large blood vessels and microvessels and endanger the heart, brain, kidney, peripheral nerves, eyes, feet and so on. According to the statistics of the World Health Organization, there are more than 100 complications of diabetes, which is the most common complication, and the incidence rate is also on the rise. The kidney plays a very important role in the body’s sugar metabolism. Glucose does not pass through the lipid bilayer of the cell membrane in the body, and must rely on the glucose transporter on the cell membrane. Sodium-coupled glucose co-transporters (SGLTs) are one of the transporters known to be responsible for the uptake of carbohydrates such as glucose. More specifically, SGLT1 is responsible for transporting glucose across the brush border membrane of the small intestine. Inhibition of SGLT1 results in a decrease in glucose absorption in the small intestine and can therefore be used in the treatment of diabetes.

Ellerelli has developed a novel SGLTs inhibitor for alternative drugs and treatments for diabetes. CN105705509 discloses the SGLTs inhibitor-pyrazole compound, which has the structure shown in the following formula (1):

It is well known for drug production process has strict requirements, the purity of pharmaceutical active ingredients will directly affect the safety and effectiveness of drug quality. Simplified synthetic route optimization, and strictly control the purity of the intermediates has a very important role in improving drug production, quality control and optimization of the dosage form development.

CN105705509 discloses a method for synthesizing a compound of the formula (1), wherein the intermediate compound 2-{(3E)-4-[3-methyl4-({5-(propyl-2-yl)) is obtained by the step B in Scheme 4. -3-[(2,3,4,6-tetra-acetyl-β-D-glucopyranosyl)oxy]-1H-pyrazol-4-yl}methyl)phenyl]but-3- Tert-butyl-1-enyl}-2,9-diazaspiro[5.5]undecane-9-carboxylate (Compound obtained in Preparation Example 12b) was obtained as a yellow foam, yield 68.6%, purity 94 %, this step involves silica gel column purification, low production efficiency, high cost, and poor quality controllability; the intermediate 2-{(3E)-4-[3-methyl 4-({5- (prop-2-yl)-3-[(2,3,4,6-tetra-acetyl-β-D-glucopyranosyl)oxy)-1H-pyrazol-4-yl}methyl) Phenyl]but-3-en-1-yl}-2,9-diazaspiro[5.5]undecane (Compound obtained in Preparation Example 18) as a yellow solid with a purity of 95.0%; The resulting intermediate compounds were all of low purity. Moreover, CN105705509 produces a compound of formula (1) having a purity of 96.7% as described in the publications of the publications 0141 and 0142. The resulting final compound is not of high purity and is not conducive to subsequent drug preparation.

Process for preparing pyranoglucose-substituted pyrazole compound, used as a pharmaceutical intermediate in SGLT inhibitor for treating diabetes.

5.00 kg of the maleate salt of the compound of the formula (16), 40 L of tetrahydrofuran, 5.47 kg of potassium phosphate and 11.67 kg of 2,3,4,6-tetra-O-pivaloyl-α-D-glucosyl bromide The compound (formula (17)) is sequentially added to the reaction vessel, heated to 40 to 45 ° C, and reacted for 4 to 5 hours, then cooled to 15 to 25 ° C, filtered, and the solid was rinsed once with tetrahydrofuran. The filter cake was dissolved in 36 L of ethyl acetate and 20 L of water and then separated. The organic phase was concentrated to ca. 18 L, 64 L acetonitrile was added and stirred for 15 h. Filtration, the filter cake was rinsed with acetonitrile, and dried under vacuum at 60 ° C for 24 h to give white crystals of the compound of formula (9c), 4.50 kg, yield 57%, HPLC purity 99.19%.

4.45 kg of the compound of the formula (9c) and 45 L of butyl acetate were sequentially added to the reaction vessel, and the temperature was lowered to 15 ° C to 20 ° C. 4.13 kg of methanesulfonic acid was added in portions and the reaction was carried out for 2 to 3 hours. 22 L of a 9% aqueous potassium hydroxide solution was added, stirred for 10 min, and the liquid phase was discarded. The organic phase was washed successively with 10 L of 9%, 4.5 L of 10% and 2 L of 2.5% aqueous potassium hydroxide and concentrated to 15 L. 68 L of n-heptane was added to the residue, and the mixture was stirred for further 12 h. Filtered and the filter cake was rinsed once with n-heptane. The solid was dried under vacuum at 60 ° C for 24 h to obtain white crystals. The methanesulfonic acid salt of the compound of formula (10c) was 4.37 kg, yield 99%, purity 97.94%.

0.73 kg of potassium hydroxide, 43 L of methanol and 4.30 kg of the compound of the formula (10c) were sequentially added to the reaction vessel, and stirred at 45 to 50 ° C for 4 hours. The temperature was lowered to 20 to 25 ° C, filtered, and 0.56 kg of acetic acid was added to the filtrate, and the mixture was stirred for 15 minutes. The reaction solution was concentrated to about 15 L under reduced pressure, and 0.40 g of acetic acid was added. After stirring for 10 min, 39 L of 3% water in ethyl acetate and 1.3 L of purified water were added dropwise. After the dropwise addition, stirring was continued for about 2 hours. Filter and filter cake was rinsed once with ethyl acetate containing 3% water. The solid was transferred to a reaction kettle, and 3.5 L of water was added and stirred for 18 h. After filtration, the filter cake was washed successively with water and an ethanol/ethyl acetate mixed solvent. The cake was vacuum dried at 35 to 40 ° C to give a white solid. Compound (1) (1), 1.84 g, yield 67%, purity 99.65%.

Fluazolepali, developed by Hengrui and Howson, is intended for the treatment of recurrent ovarian cancer, triple-negative breast cancer, advanced gastric cancer and other advanced solid tumors. Currently, the drug has been introduced into China for recurrent ovarian cancer. Clinical stage.

In February 2019, a randomized, double-blind, controlled, multicenter, phase III clinical study (CTR20190294) of flazopril capsule versus placebo for maintenance of recurrent ovarian cancer was initiated in China and was sponsored by Hengrui Medicine.

Jiangsu Hansoh Pharmaceutical , in collaboration with Jiangsu Hengrui Medicine , is developing an oral capsule formulation of fluazolepali (fluzoparib; SHR-3162), a small molecule inhibitor to PARP-1 and PARP-2, for the treatment of solid tumors including epithelial ovarian, fallopian tube or primary peritoneal, breast and gastric cancer.

Process for preparing heterocyclic compounds (presumed to be fluazolepali ) and its intermediates as PARP inhibitors useful for treating cancer.

Example 1

The compound and 5.0kg of 10% palladium on carbon 250g, 80L of methanol was added to the kettle at 0.4MPa, 24h 25 ℃ hydrogenation reaction. The palladium carbon was removed by filtration, the filter cake was washed with methanol, and the filtrate was collected, evaporated to dryness under reduced pressure, and ethyl acetate (20 L) was added to the concentrate, and the mixture was stirred and evaporated, and then cooled to 0° C. ～3, stirring, filtration, filter cake and then adding 20 L of ethyl acetate, pulping at room temperature for 3 to 4 h, filtration, vacuum drying at 45 ° C for 6-8 h to obtain 5.5 kg of compound 3 solid, yield 91.7%, HPLC purity 99.69%.

Example 2

According to the method of Example 19 of CN102686591A, 2 g of the compound 3 and 2.79 g of the compound 4 were charged to obtain 3.6 g of the compound of the formula I in a yield of 87.8%.

Example 3

At room temperature, 2.0 g of compound 2 (prepared according to the method disclosed in WO2009025784) was dissolved in 30 mL of isopropanol, and concentrated sulfuric acid was added dropwise with stirring to adjust the pH to 3, and stirred at room temperature without solid precipitation; the reaction solution was poured into 150 mL of n-hexane. After stirring at room temperature, no solid precipitated, and the sulfate solid of Compound 2 could not be obtained.

At room temperature, 1.28 g of compound 2 was dissolved in 10 mL of isopropanol, and 20% acetic acid/isopropanol solution was added dropwise with stirring to adjust the pH to 3, and stirred at room temperature without solid precipitation; the reaction solution was poured into 100 mL of n-hexane, and continued. After stirring at room temperature, no solid precipitated, and the acetate solid of Compound 2 could not be obtained.

Example 6

1.05g of compound 2 was dissolved in 10mL of isopropanol at room temperature, and the pH was adjusted to 3 by adding 15% citric acid/isopropanol solution while stirring. At room temperature, no solid precipitated; the reaction solution was poured into 100 mL of n-hexane. After stirring at room temperature, no solid precipitated, and the citrate solid of Compound 2 could not be obtained.

Example 7

1.12 g of compound 2 was dissolved in 10 mL of isopropanol at room temperature, and 0.74 g of maleic acid was added thereto with stirring. The mixture was stirred at room temperature, filtered, and the filter cake was washed with isopropyl alcohol and dried in vacuo to obtain the maleate salt of compound 2. 1.51 g, yield 84.6%.

The fibroblast growth factor receptor (FGFR) belongs to the receptor tyrosine kinase transmembrane receptor and includes four receptor subtypes, namely FGFR1, FGFR2, FGFR3 and FGFR4. FGFR regulates various functions such as cell proliferation, survival, differentiation and migration, and plays an important role in human development and adult body functions. FGFR is abnormal in a variety of human tumors, including gene amplification, mutation and overexpression, and is an important target for tumor-targeted therapeutic research.

FGFR4, a member of the FGFR receptor family, forms dimers on the cell membrane by binding to its ligand, fibroblast growth factor 19 (FGF19), and the formation of these dimers can cause critical tyrosine in FGFR4’s own cells. The phosphorylation of the amino acid residue activates multiple downstream signaling pathways in the cell, and these intracellular signaling pathways play an important role in cell proliferation, survival, and anti-apoptosis. FGFR4 is overexpressed in many cancers and is a predictor of malignant invasion of tumors. Decreasing and reducing FGFR4 expression can reduce cell proliferation and promote apoptosis. Recently, more and more studies have shown that about one-third of liver cancer patients with continuous activation of FGF19/FGFR4 signaling pathway are the main carcinogenic factors leading to liver cancer in this part of patients. At the same time, FGFR4 expression or high expression is also closely related to many other tumors, such as gastric cancer, prostate cancer, skin cancer, ovarian cancer, lung cancer, breast cancer, colon cancer and the like.

The incidence of liver cancer ranks first in the world in China, with new and dead patients accounting for about half of the total number of liver cancers worldwide each year. At present, the incidence of liver cancer in China is about 28.7/100,000. In 2012, there were 394,770 new cases, which became the third most serious malignant tumor after gastric cancer and lung cancer. The onset of primary liver cancer is a multi-factor, multi-step complex process with strong invasiveness and poor prognosis. Surgical treatments such as hepatectomy and liver transplantation can improve the survival rate of some patients, but only limited patients can undergo surgery, and most patients have a poor prognosis due to recurrence and metastasis after surgery. Sorafenib is the only liver cancer treatment drug approved on the market. It can only prolong the overall survival period of about 3 months, and the treatment effect is not satisfactory. Therefore, it is urgent to develop a liver cancer system treatment drug targeting new molecules. FGFR4 is a major carcinogenic factor in liver cancer, and its development of small molecule inhibitors has great clinical application potential.

At present, some FGFR inhibitors have entered the clinical research stage as anti-tumor drugs, but these are mainly inhibitors of FGFR1, 2 and 3, and the inhibition of FGFR4 activity is weak, and the inhibition of FGFR1-3 has hyperphosphatemia. Such as target related side effects. Highly selective inhibitor of FGFR4 can effectively treat cancer diseases caused by abnormal FGFR4 signaling pathway, and can avoid the side effects of hyperphosphatemia caused by FGFR1-3 inhibition. Highly selective small molecule inhibitors against FGFR4 in tumor targeted therapy The field has significant application prospects.

SYN

PATENT

WO2017198149

where it is claimed to be an FGFR-4 inhibitor for treating liver and prostate cancers, assigned to Jiangsu Hansoh Pharmaceutical Group Co Ltd and Shanghai Hansoh Biomedical Co Ltd .

PATENT

WO2019085860

Compound (R)-N-(5-Cyano-4-((1-methoxypropan-2-yl)amino)pyridin-2-yl)-7-formyl-6-((2-carbonyl-) 1,3-oxazepine-3-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxamide (shown as Formula I). The compound of formula (I) is disclosed in Hausen Patent PCT/CN2017/084564, the compound of formula I is a fibroblast growth factor receptor inhibitor, and the fibroblast growth factor receptor (FGFR) belongs to the receptor tyrosine kinase transmembrane receptor. The body includes four receptor subtypes, namely FGFR1, FGFR2, FGFR3 and FGFR4. FGFR regulates various functions such as cell proliferation, survival, differentiation and migration, and plays an important role in human development and adult body functions. FGFR is abnormal in a variety of human tumors, including gene amplification, mutation and overexpression, and is an important target for tumor-targeted therapeutic research.

The second step is 3-((2-(dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)-1,3- Preparation of oxazepine-2 ketone

[0054]

[0055]

4-(((2-(Dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)amino) in an ice water bath Butan-1-ol (0.6 g, 1.94 mmol) was dissolved in DCE (15 mL), then bis(trichloromethyl) carbonate (0.22 g, 0.76 mmol) was added and triethylamine (0.78 g, 7.76) was slowly added dropwise. Methyl) and then stirred at room temperature for 3 hours. The reaction temperature was raised to 80 ° C, and the reaction was carried out at 80 ° C for 6 hours. After the reaction was cooled to room temperature, it was diluted with CH 2 Cl 2 (100 mL), and the organic phase was washed sequentially with water (10 mL) and brine (15 mL) Drying with sodium sulfate, concentration and column chromatography to give the compound 3-((2-(dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl) )methyl)-1,3-oxazepin-2-one (0.37 g, 57%).

[0056]

MS m/z (ESI): 336.2 [M+H] + .

[0057]

The third step is phenyl 7-(dimethoxymethyl)-6-((2-carbonyl-1,3-oxazepine-3-yl)methyl)-3,4-dihydro-1, Preparation of 8-naphthyridin-1(2H)-carboxylate

The second step is 3-((2-(dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)-1,3- Preparation of oxazepine-2 ketone

4-(((2-(Dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)amino) in an ice water bath Butan-1-ol (0.6 g, 1.94 mmol) was dissolved in DCE (15 mL), then bis(trichloromethyl) carbonate (0.22 g, 0.76 mmol) was added and triethylamine (0.78 g, 7.76) was slowly added dropwise. Methyl) and then stirred at room temperature for 3 hours. The reaction temperature was raised to 80 ° C, and the reaction was carried out at 80 ° C for 6 hours. After the reaction was cooled to room temperature, it was diluted with CH 2 Cl 2 (100 mL), and the organic phase was washed sequentially with water (10 mL) and brine (15 mL) Drying with sodium sulfate, concentration and column chromatography to give the compound 3-((2-(dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl) )methyl)-1,3-oxazepin-2-one (0.37 g, 57%).

MS m/z (ESI): 336.2 [M+H] + .

The third step is phenyl 7-(dimethoxymethyl)-6-((2-carbonyl-1,3-oxazepine-3-yl)methyl)-3,4-dihydro-1, Preparation of 8-naphthyridin-1(2H)-carboxylate

The compound was originally claimed in WO2017202291 , covering thiophene derivative URAT1 inhibitors, useful for treating hyperuricemia and gouty arthritis, assigned to Medshine Discovery Inc , but naming the inventors.and has been reported in some instances to be a URAT1 modulator. In June 2018, an IND application was filed in

Uric acid is a product of the metabolism of terpenoids in animals. For humans, due to the lack of uric acid enzymes that continue to oxidatively degrade uric acid, uric acid is excreted in the human body as the final product of sputum metabolism through the intestines and kidneys. Renal excretion is the main pathway for uric acid excretion in humans. The upper limit of the normal range of uric acid concentration in the human body is: male 400 μmol/L (6.8 mg/dL) and female 360 μmol/L (6 mg/dL). Abnormal uric acid levels in the human body are often due to an increase in uric acid production or a decrease in uric acid excretion. Conditions associated with abnormal levels of uric acid include hyperuricemia, gout, and the like.

Hyperuricemia refers to a disorder in which the metabolism of substances in the human body is disordered, resulting in an increase or decrease in the synthesis of uric acid in the human body, and an abnormally high level of uric acid in the blood. Gouty arthritis refers to the fact that when uric acid is more than 7 mg/dL in human blood, uric acid is deposited as a monosodium salt in the joints, cartilage and kidneys, causing excessive reaction (sensitivity) to the body’s immune system and causing painful inflammation. The general site of attack is the big toe joint, ankle joint, knee joint and so on. Red, swollen, hot, and severe pain in the site of acute gout attacks, usually in the midnight episode, can make people wake up from sleep. In the early stages of gout, the attack is more common in the joints of the lower extremities. Hyperuricemia is the pathological basis of gouty arthritis. The use of drugs to lower blood uric acid concentration is one of the commonly used methods to prevent gouty arthritis.

In Europe and the United States, the onset of hyperuricemia and gout disease is on the rise. Epidemiological studies have shown that the incidence of gouty arthritis accounts for 1-2% of the total population and is the most important type of arthritis in adult males. Bloomberg estimates that there will be 17.7 million gout patients in 2021. In China, the survey showed that among the population aged 20 to 74, 25.3% of the population had a high blood uric acid content and 0.36% had gout disease. At present, clinical treatment drugs mainly include 1) inhibition of uric acid-producing drugs, such as xanthine oxidase inhibitor allopurinol and febuxostat; 2) uric acid excretion drugs, such as probenecid and benzbromarone; 3) Inflammation inhibitors, such as colchicine. These drugs have certain defects in treatment, poor efficacy, large side effects, and high cost are some of the main bottlenecks in their clinical application. It has been reported that 40%-70% of patients with serum uric acid levels do not meet the expected therapeutic goals (<6mg/dL) after receiving standard treatment.

As a uric acid excretion agent, its mechanism of action is to reduce the reabsorption of uric acid by inhibiting the URAT1 transporter on the brush-like edge membrane of the proximal convoluted tubule. Uric acid is a metabolite of sputum in the body. It is mainly filtered by glomerulus in the original form, reabsorbed and re-secreted by the renal tubules, and finally excreted through the urine. Very few parts can be secreted into the intestinal lumen by mesenteric cells. The S1 segment of the proximal convoluted tubule is a site of uric acid reabsorption, and 98% to 100% of the filtered uric acid enters the epithelial cells through the uric acid transporter URAT1 and the organic anion transporter OAT4 on the brush epithelial cell border of the tubular epithelial cells. The uric acid entering the epithelial cells is reabsorbed into the capillaries around the tubules via the renal tubular basement membrane. The S2 segment of the proximal convoluted tubule is the site of re-secretion of uric acid, and the amount secreted is about 50% of the excess of the small filter. The uric acid in the renal interstitial enters the epithelial cells first through the anion transporters OAT1 and OAT3 on the basal membrane of the tubular epithelial cells. The uric acid entering the epithelial cells passes through another anion transporter MRP4 on the brush border membrane and is discharged into the small lumen. The S3 segment of the proximal convoluted tubule may be a reabsorption site after uric acid secretion, and the amount of reabsorption is about 40% of the excess of the microsphere filtration, and similar to the first step of reabsorption, URAT1 may be a key reabsorption transporter. Therefore, if the urate transporter URAT1 can be significantly inhibited, it will enhance the excretion of uric acid in the body, thereby lowering blood uric acid level and reducing the possibility of gout attack.

In December 2015, the US FDA approved the first URAT1 inhibitor, Zurampic (Leinurad). The 200 mg dose was approved in combination with xanthine oxidase inhibitor XOI (such as Febuxostat, etc.) for the treatment of hyperuricemia and gouty arthritis, but the combination was compared with the xanthine oxidase inhibitor alone. The effect is not very significant. The Zurampic 400 mg dose was not approved due to significant toxic side effects at high doses (the incidence of renal-related adverse events, especially the incidence of kidney stones). Therefore, the FDA requires the Zurampic label to be filled with a black box warning to warn medical staff Zulampic of the risk of acute kidney failure, especially if it is not used in conjunction with XOI. If the over-approved dose uses Zurampic, the risk of kidney failure is even greater. high. At the same time, after the FDA asked for the listing of Zurampic, AstraZeneca continued its investigation of kidney and cardiovascular safety. Therefore, the development of a new type of safe blood-supplemented uric acid drug has become a strong demand in this field.

Novel crystalline forms of URAT1 inhibitor (designated as Forms A and B) are claimed. The compounds are disclosed to be useful for treating hyperuricemia and gouty arthritis.

Novel crystalline forms of a URAT1 inhibitor, designated as Forms A and B, and their preparation.

Example 1: Preparation of a compound of formula (I)

synthetic route:

Step 1: Synthesis of Compound 2

In a three-necked flask (10 L), 4.5 L of dimethyl sulfoxide was added, and potassium t-butoxide (836.66 g, 7.46 mol, 2 eq) was added with stirring, and stirring was continued for 10 minutes until the dissolution was clear, and then cooled to an ice water bath. The internal temperature of the reaction solution was 20-25 °C. To the above solution, a solution of Compound 1 (500.05 g, 3.73 mol, 1 eq) in dimethyl sulfoxide (500 mL) was added dropwise, and the mixture was stirred for 30 minutes, and then carbon disulfide (283.86 g, 3.73 mol, 1 eq) was added dropwise thereto. ), after the completion of the dropwise addition, the reaction was stirred for 30 minutes. Further, ethyl bromoacetate (1250 g, 7.46 mol, 2 eq) was added dropwise thereto, and the mixture was stirred for further 2 hours. Finally, potassium carbonate (515.52 g, 7.46 mol, 1 eq) was added, and the temperature was raised to an internal temperature of 65 ° C, and the reaction was further stirred for 8 hours. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was diluted with ethyl acetate (10 L), and then 1M hydrochloric acid (2 L) and water (2 L) were added and stirred for 10 minutes, and the mixture was allowed to stand. The aqueous layer was separated and the organic phase was washed with water (2L×3). The combined aqueous layers were extracted with ethyl acetate (3L). All organic phases were combined and washed with saturated brine (2 L×2). The organic phase was dried over an appropriate amount of anhydrous sodium sulfate, and then filtered, and then evaporated. On the same scale, 6 batches were fed in parallel, and the combined black and red oily products were obtained. After the crude product was allowed to stand for 72 hours, a large amount of solid was precipitated, ethanol (2 L) was added thereto, stirred for 30 minutes, filtered, and the cake was collected and dried in vacuo to give Compound 2. . 1 H NMR (400 MHz, CDCl3 . 3 ) [delta]: 4.32 (Q, J = 7.2 Hz, 2H), 4.19 (Q, J = 7.2 Hz, 2H), 3.56 (S, 2H), 3.25 (T, J = 6.8Hz , 2H), 3.19 (t, J = 14.4 Hz, 2H), 2.26-2.17 (m, 2H), 1.37 (t, J = 7.2 Hz, 3H), 1.27 (t, J = 7.2 Hz, 3H); MS m/z = 364.8 [M+H] + .

Compound 3 (406.2 g, 1.65 mol, 1 eq) was dissolved in acetonitrile (6 L), then N-bromosuccinimide (1484.2 g, 6.60 mol, 4 eq) was slowly added, and the obtained reaction mixture was at 23 to 25 ° C. The reaction was stirred for 12 hours. After the reaction was completed, the reaction liquid was concentrated to about 1.0 L. The solid was removed by filtration, and a saturated solution of sodium hydrogensulfite (1 L) was added to the filtrate and stirred for 10 min. Add acid ethyl ester and extract three times, 2L each time. The organic phases were combined and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. Petroleum ether (3 L) was added to the residue, and the mixture was stirred at 30 ° C for 30 minutes. After filtration, the filter cake was washed 5 times with petroleum ether, 200 mL each time, until no product remained in the filter cake. Combine all the organic phases and spin dry to obtain a crude product. Petroleum ether (100 mL) was added to the crude product, stirred well, filtered, and filtered, and then dried in vacuo. . 1 H NMR (400 MHz, CDCl3 . 3) [delta]: 4.24 (Q, J = 7.2 Hz, 2H), 3.19 (T, J = 6.8Hz, 2H), 2.95 (T, J = 14.4Hz, 2H), 2.17-2.07 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H).

In a 3 L three-necked flask, Compound 8 (301.25 g) was added to tetrahydrofuran (600 mL), and the mixture was cooled to an internal temperature of 0 to 10 ° C under ice-cooling. Diisopropylethylamine (635.72 g) was added dropwise, and after completion of the dropwise addition, the ice water bath was removed, and the mixture was stirred at an internal temperature of 10 to 15 ° C for about 10 minutes. Filter and filter cake was washed with tetrahydrofuran (100 mL x 2). The filtrates were combined to give a solution A for use.

Tetrahydrofuran (2 L) was added to a 5 L reaction flask containing thiophosgene (257.48 g). The mixture was stirred and cooled to an internal temperature of 0 to 10 ° C in an ice water bath, and the solution A was slowly added dropwise thereto, and the dropwise addition was completed within about 5.5 hours, and stirring was continued for 10 minutes. After the reaction was completed, it was filtered, and the filter cake was washed with tetrahydrofuran (150 mL × 2). The filtrate was combined and concentrated under reduced pressure to remove solvent. Tetrahydrofuran (400 mL) was added to the residue, which was dissolved to give a solution B.

The hydrazine hydrate (112.94 g) was added to tetrahydrofuran (2.5 L), and the mixture was cooled to an internal temperature of 5 to 10 ° C under ice-cooling. Solution B was added dropwise, and the addition was completed for about 2 hours, and stirring was continued for 10 minutes. After the reaction was completed, the reaction was stopped. The ice water bath was removed, N,N-dimethylformamide dimethyl acetal (333.45 g) was added, and the mixture was heated to an internal temperature of 60 to 65 ° C, and the reaction was stopped after the heat retention reaction for 3 hours.

Tetrahydrofuran (1.2 L) was added to a 5 L reaction flask, and Compound 11 (243.03 g) was added with stirring. After the solution was dissolved, pure water (1.2 L) was added, and then lithium hydroxide monohydrate (125.46 g) was added, and the mixture was stirred at 20 to 25 ° C for about 2.5 hours. After the reaction was completed, the reaction was stopped. The reaction solution was concentrated under reduced pressure at 40 ° C to remove organic solvent. Pure water (1 L) was added to the residue, and the mixture was extracted with t-butyl methyl ether (300 mL). The aqueous phase was placed in a 10 L three-necked flask and cooled to 5 to 10 ° C in an ice bath. The pH was adjusted to 2 to 3 with a 40% hydrobromic acid solution, and a large amount of a pale yellow solid precipitated. Stirring was continued for 30 minutes, and the pH was again measured to be 2-3. Stirring was continued for 20 minutes and filtered. The filter cake was washed with pure water (150 mL x 3). The filter cake was collected, pure water (1500 mL) was added, and the mixture was beaten at room temperature for 1 hour. After filtration, the filter cake was washed with pure water (150 mL × 2), and the filter cake was collected and dried under vacuum at 40 ° C for 3 hours to obtain a compound of the formula (I). . 1 H NMR (400 MHz, the CD . 3 the OD) [delta]: 3.27 (T, J = 15.6Hz, 2H), 2.60-2.47 (m, 2H), 2.27-2.17 (m, 2H), 2.10-2.03 (m, IH) , 1.68 (d, J = 1.2 Hz, 6H), 1.15.10.10 (m, 2H), 0.80-0.71 (m, 2H); MS m/z = 477.99 [M+H] + , 480.1 [M+H+ 2] + .

Example 2: Preparation of Form A of Compound of Formula (I)

The compound of the formula (I) (50 mg) was added to a glass bottle, and methanol (0.4 mL) was added thereto, followed by stirring to a suspension or a solution. The suspension sample was placed in a thermomixer (40 ° C), shaken at 40 ° C for 60 hours, and then centrifuged to collect a sample. The above-mentioned lysed sample was volatilized at room temperature, centrifuged, and the sample was collected. The above sample was dried in a vacuum oven (40 ° C) overnight, and its crystalline form was examined by XRPD to obtain a crystal form of the final product having a crystalline form of the compound of the formula (I).

The compound of the formula (I) (50 mg) was added to a glass bottle, and ethyl acetate (0.4 mL) was added and stirred to a suspension or a solution. The suspension sample was placed in a thermomixer (40 ° C), shaken at 40 ° C for 60 hours, and then centrifuged to collect a sample. The above-mentioned lysed sample was volatilized at room temperature, centrifuged, and the sample was collected. The above sample was dried in a vacuum oven (40 ° C) overnight, and its crystalline form was examined by XRPD to obtain a crystal form of the final product having a crystalline form of the compound of the formula (I).

Example 3: Preparation of Form B of Compound of Formula (I)

The compound of the formula (I) (50 mg) was added to a glass bottle, tetrahydrofuran (0.4 mL) was added, and the mixture was stirred to dissolve. The above-mentioned lysed sample was volatilized at room temperature, centrifuged, and the sample was collected. The collected sample was dried in a vacuum oven (40 ° C) overnight, and its crystalline form was examined by XRPD to obtain a crystalline form of the final product in the form of Form B of the compound of formula (I).

Example 4: Solubility test of Form A of the compound of formula (I)

1. Preparation of diluent and mobile phase

Diluent: Accurately measure 300mL of pure water and 100mL of pure acetonitrile, mix in a 1L glass bottle, ultrasonic degassing for 10 minutes and then set aside.

2. Preparation of the reference solution (using the A crystal form itself as a control sample)

Accurately weigh 5 mg of Form A, place it in a sample vial, add 10 mL of diluent, sonicate for 5 minutes, then cool to room temperature and mix well, and mark it as working reference solution STD-1.

Accurately weigh 5 mg of Form A, place it in a sample vial, add 10 mL of diluent, sonicate for 5 minutes, then cool to room temperature and mix well, and mark it as working reference solution STD-2.

3. Preparation of linear solution

The above working reference solution STD-1 was diluted 1 time, 10 times, 100 times, 1000 times and 2000 times, and recorded as linear solutions L1, L2, L3, L4 and L5.

4. Solubility test

Accurately weigh 6mg of A crystal form into 8mL glass bottle, then accurately add 3mL different solvent (0.1N hydrochloric acid solution, 0.01N hydrochloric acid solution, purified water, pH3.8 buffer solution, pH4.5 buffer solution, pH5 .5 buffer solution, pH 6.0 buffer solution, pH 7.4 buffer solution, pH 6.8 buffer solution), made into a suspension. A stir bar was added to the above suspension, and the mixture was thoroughly stirred at 37 ° C in the dark. After stirring, the solids in the pH 7.4 buffer solution and the pH 6.8 buffer solution were all dissolved, and 6 mg of the A crystal form was accurately weighed, added to the buffer solution, and thoroughly stirred again to prepare a suspension. After stirring for 4 hours and 24 hours, the sample was centrifuged, and the solution was filtered through a filter and the concentration thereof was measured by HPLC. The HPLC analysis method is shown in Table 3.

The compound was originally claimed in WO2013152135 , and may provide the structure of TL-487 , a small molecule inhibitor to HERs, being investigated by Teligene for the treatment of breast cancer; in July 2016, the company intended to develop the product as a class 1.1 chemical drug in China.

Novel crystalline maleate salt of (E)-N-(3-cyano-7-ethoxy-4-((4-phenoxyphenyl)amino)quinolin-6-yl)-4-(dimethylamino)but-2-enamide (first disclosed in WO2013152135) and its hydrates (monohydrate) and anhydrates, process for its preparation, composition comprising it and its use for treating cancers such as breast cancer, ovary cancer, colon cancer, prostate cancer, kidney cancer, bladder cancer, stomach cancer, lung cancer, mantle cell lymphoma and multiple myeloma are claimed. The compound is disclosed to be an irreversible inhibitor to BTK and Her-2 (also known as Erb-2 or neu).

(E) -N- (3-cyano-7-ethoxy-4- ( (4-phenoxyphenyl) amino) quinolin-6-yl) -4- (dimethylamino) but-2-enamide is mentioned in WO2013152135 and corresponds to the compound of the Formula I:

Formula I

Compounds derived from 3-cyanoquinoline have been shown to have anti-tumor activity, which may make them useful as chemotherapeutic agents in treating various cancers, including but not limited to, pancreatic cancer, melanoma, lymphatic cancer, parotid tumors, Barrett’s esophagus, esophageal carcinomas, head and neck tumors, ovarian cancer, breast cancer, epidermoid tumors, cancers of major organs, such as kidney, bladder, larynx, stomach, and lung, colonic polyps and colorectal cancer and prostate cancer. Examples of compounds derived from 3-cyanoquinoline are disclosed and shown to possess anti-tumor activity in many literatures. One limitation of certain 3-cyanoquinoline compounds is that they are not water soluble in a free base form.

The crystalline form of a particular drug as a salt, a hydrate and/or any polymorph thereof is often one important determinant of the drug’s ease of preparation, stability, water solubility, storage stability, ease of formulation and in-vivo pharmacology. It is possible that one crystalline form is preferable over another where certain aspects such as ease of preparation, stability, water solubility and/or superior pharmacokinetics are deemed to be critical. Crystalline forms of (E) -N- (3-cyano-7-ethoxy-4- ( (4-phenoxyphenyl) amino) quinolin-6-yl) -4- (dimethylamino) but-2-enamide salts that possess a higher degree of water solubility than the free base but are stable fulfill an unmet need for stable, crystalline, water-solubl

95%ethanol (4.0 ml) was added to (E) -N- (3-cyano-7-ethoxy-4- ( (4-phenoxyphenyl) amino) quinolin-6-yl) -4- (dimethylamino) but-2-enamide (500 mg, 0.99 mmol, 1.0 eq) , followed sulfuric acid (101.9 mg, 1.04 mmol, 1.05 eq) in 95%ethanol (1.0 ml) was added dropwise to the reaction mixture. Then an amount of precipitate was founded. Another 95% (60 ml) was added to the reaction mixture and the reaction mixture was heated to 70℃. Filtered and the filtrate was heated to 70℃ again. Then the reaction mixture was cooled to room temperature and The reaction mixture was crystallized at -10℃ for 41.5h. Filtered the precipitated solid and dried at 40℃ under vacuum for 1 hour to get the title compound (260 mg) as a yellow solid.

X-ray detection shows an amorphous structure to the compound as FIG. 9.

(E) -N- (3-cyano-7-ethoxy-4- ( (4-phenoxyphenyl) amino) quinolin-6-yl) -4- (dimethylamino) but-2-enamide (500 mg, 0.99 mmol, 1.0 eq) , L-malic acid (139.4 mg, 1.04 mmol, 1.05 eq) and 95%ethanol (5.0 ml) was added to a 50 ml round-bottom flask. The reaction mixture was heated to 70℃. Filtered and the filtrate was crystallized under -10℃ for 45.5h. A little of precipitate was founded and then the reaction mixture was evaporated under vacuum at 40℃ to give the target (370 mg) as a yellow solid.

X-ray detection shows an amorphous structure to the compound in FIG. 8

To a solution of (E) -N- (3-cyano-7-ethoxy-4- ( (4-phenoxyphenyl) amino) quinolin-6-yl) -4- (dimethylamino) but-2-enamide (500 mg, 0.99 mmol, 1.0 eq) , citric acid (198.8 mg, 1.04 mmol, 1.05 eq) and 95%ethanol (5.0 ml) . The reaction mixture was heated to 70℃. Filtered and the filtrate was crystallized under -10℃ for 45h. A little of precipitate was founded and then the reaction mixture was evaporated under vacuum at 40℃ to give the target compound (610 mg) as a yellow solid.

X-ray detection shows an crystalline structure to the compound in FIG. 7.

(E) -N- (3-cyano-7-ethoxy-4- ( (4-phenoxyphenyl) amino) quinolin-6-yl) -4- (dimethylamino) but-2-enamide free base (0.091 kg) is rinsed with a 10%solution of USP purified water in n-propanol (0.082 kg, 0.10 L) followed by the addition of water: n-propanol solution (0.74 kg, 0.90 L) . Maleic acid is added (1.01 equiv) and the mixture is rinsed with 10%water: n-propanol (0.082 kg, 0.10 L) . The mixture is quickly heated to 50-60 ℃ and held for a minimum of 15 min. until a solution is obtained. The hot solution is clarified through a pre-heated 50-60 ℃, 0.2 Mm filter cartridge and the filtrates are collected in a preheated 45-55℃, 2 L multi-neck flask. The filter cartridge is rinsed through with 10%water: n-propanol pre-heated to 45-55 ℃ (0.082 kg, 0.10 L) . The solution is cooled over at least one hour to 40 ℃ and held at that temperature for 12 hours then cooled to room temperature (25 ℃) over a minimum of four hours and held at that temperature for at least two hours. The mixture is filtered on a 12.5 cm diameter Buchner funnel for 5 min., then rinsed and washed with prefiltered10%water: n-propanol solution (2 x 0.12 kg, 2 x 0.15 L) . The cake is dammed and suction maintained until dripping essentially stops, about 1 h.

To a solution of (E) -N- (3-cyano-7-ethoxy-4- ( (4-phenoxyphenyl) amino) quinolin-6-yl) -4- (dimethylamino) but-2-enamide (21.5 g, 42.4 mmol, 1.0 eq) and ethanol (300 ml) . maleic acid (5.2 g, 44.8 mmol, 1.05 eq) was added to the reaction mixture. An amount of precipitate was founded, then the reaction mixture was heated to 70 ℃. Another ethanol (1980 ml) was added to the reaction mixture in several times and the reaction temperature was keep at 70 ℃. Filtered and filtrate was cooled to room temperature, stop stirring and stand for 16-20 hours. Filtered and the solid was dried at room temperature for 24 hours to get the title compound.

Natural products have historically been, and continue to be, an invaluable source for the discovery of various therapeutic agents. Oridonin, a natural diterpenoid widely applied in traditional Chinese medicines, exhibits a broad range of biological effects including anticancer and anti-inflammatory activities. To further improve its potency, aqueous solubility and bioavailability, the oridonin template serves as an exciting platform for drug discovery to yield better candidates with unique targets and enhanced drug properties. A number of oridonin derivatives (e.g. HAO472) have been designed and synthesized, and have contributed to substantial progress in the identification of new agents and relevant molecular mechanistic studies toward the treatment of human cancers and other diseases. This review summarizes the recent advances in medicinal chemistry on the explorations of novel oridonin analogues as potential anticancer therapeutics, and provides a detailed discussion of future directions for the development and progression of this class of molecules into the clinic.

ERROR IN STRUCTURE

As described for Example 2 according to the patent ZL03139760.3 obtained chidamide poor purity (about 95%).LC / MS analysis results shown in Figure 1, show that the product contains N- (2- amino-5-fluorophenyl) -4- (N- (3- pyridin-acryloyl group of 4.7% of the structure shown in formula II) aminomethyl) benzamide.1H NMR analysis of the results shown in Figure 2, show that the product contains 1.80% of tetrahydrofuran, far beyond the technical requirements for people with drug registration International Conference on Harmonization (ICH, International Conference of Harmonizition) provided 0.072% residual solvent limits.Therefore, the solid

December 2010: of PTCL by a conventional phase II directly into Phase II clinical trial registered drug trial center and by recognition

September 2012: of PTCL indication test deadline

December 2012: of PTCL clinical summary will be held

January 2013: Chidamide declare China NDA

December 2014: the State Food and Drug Administration (CFDA) approved the listing

Chidamide overview, location and clinical significance

Chidamide (Chidamide, love spectrum sand ® / Epidaza®) Shenzhen microchip biotechnology limited liability company developed a new subtype selective histone having a chemical structure and is eligible for a global patent licensing deacetylase inhibitor, belong to the new mechanisms of epigenetic regulation new class of targeted anticancer drugs, has now completed with relapsed or refractory peripheral T-cell lymphoma clinical trial study registered indications, was in March 2013 to the SFDA reporting new drug certificate (NDA) and the marketing authorization (MAA). While a number of Chinese Cancer clinical trials undertaken Chidamide is also China’s first approved by the US FDA clinical studies in the United States of Chinese chemical original new drug trials in the United States Phase I has been completed. Chidamide has won the national “Eleventh Five-Year” 863 major projects (project number: 2006AA020603) and the national “Eleventh Five-Year”, “significant Drug Discovery” science and technology and other major projects funded project (project number: 2009ZX09401-003), was chosen the Ministry of Science and one of the “Eleventh five-Year” major national scientific and technological achievements.

Relapsed or refractory peripheral T-cell lymphoma (PTCL) is Chidamide first approvedclinical indications, PTCL belongs to the category of rare diseases, the lack of standard drug currently recommended clinical treatment, conventional chemotherapy response rate is low, recur, 5-year overall survival rate was about 25%. The world’s first PTCL treatment Folotyn (intravenous drug use) is eligible for FDA clearance to market in 2009, the second drugs Istodax (intravenous drug use) approved by the FDA in 2011. Add a new drug information for these drugs is very expensive, and were listed in China. Chidamide album clinical trial results showed that the primary endpoint of objective response rate was 28%, reaching the intended target research and development; sustained remission rate of 24% three months; drug safety was significantly better than the international similar drugs, and oral medication.
Chidamide is a completely independent intellectual property rights China originator of innovative medicines, has been multi-national patent. In China, for patients with relapsed or refractory PTCL to carry out effective drug treatment is urgent clinical need, Chidamide expected to bring new treatment options for patients with PTCL, prolong survival and improve quality of life of patients.

In China, for the effective treatment of patients with relapsed or refractory PTCL has undertaken urgent clinical need

Chidamide (Chidamide) has been multi-national invention patents

In October 2006, the US HUYA biological microchip company formally signed the International Patent Chidamide licensing and international clinical cooperative development agreement; the United States in the ongoing Phase I clinical

Chidamide (Epidaza), a class I HDAC inhibitor, was discovered and developed by ChipScreen and approved by the CFDA in December 2014 for the treatment of recurrent of refractory peripheral T-cell lymphoma. Chidamide, also known as CS055 and HBI- 8000, is an orally bioavailable benzamide type inhibitor of HDAC isoenzymes class I , as well as class IIb 10, with potential antineoplastic activity. It selectively binds to and inhibits HDAC, leading to an increase in acetylation levels of histone protein H3.

Chidamide, the English called Chidamide, by the Shenzhen-core biotechnology limited liability company independent design and synthesis of a novel anti-cancer drugs with new chemical structures and global intellectual property, and its chemical name N- (2-amino-_4_ fluorophenyl) -4_ (N- (3- topiramate Li acryloyl) aminomethyl) benzamide, its chemical structure of the structural formula I

The patent ZL03139760.3 and said US7,244,751, Chidamide have histone deacetylase inhibitory activity can be used to treat the differentiation and proliferation-related diseases such as cancer and psoriasis, especially for leukemia and solid tumors with excellent results.

Patent No. ZL03139760.3 and US7,244,751 discloses a method for preparing chidamide, but did not specify whether the resulting product is a crystalline material, nor did the presence or absence of the compound polymorphism.In the above patent, the activity of the compound for evaluation is not conducted in a solid state and, therefore, does not disclose any description about characteristics of the crystal.

Chipscreen grabs CFDA approval for chidamide

Chipscreen BioSciences announced that the CFDA had approved chidamide for the treatment of relapsed or refractory peripheral T-cell lymphoma (PTCL) in December 2014. The drug and Hengrui’s apatinib were the only two NCEs launched by domestic drug makers last year.

Chidamide (CS055/HBI-8000) is a HDAC1/2/3/10 inhibitor derived from entinostat (MS-27-275)[1] which was first discoved by Mitsui Pharmaceuticals in 1999. Chipscreen holds worldwide IP rights to chidamide (patents: WO2004071400, WO2014082354).

Syndax Pharmaceuticals (NASDAQ: SNDX) is testing entinostat in breast cancer and NSCLC in pivotal trials. The FDA granted Breakthrough Therapy Designation to entinostat for advanced breast cancer in 2013. Eddingpharm in-licensed China rights to entinostat from Syndax in September 2013.

The FDA has approved three HDAC inhibitors, known as Zolinza (vorinostat), Istodax (romidepsin) and Beleodaq (belinostat), for the treatment of PTCL. Celgene priced Istodax at $12000-18000/month and reported annual sales of $54 million in 2013. The efficacy and safety profile of chidamide compares favorably with romidepsin.

Although a dozen of companies are developing generic vorinostat and romidepsin, no chemical 3.1 NDA has been submitted to the CFDA so far. Chipscreen will be the only domestic maker of HDAC inhibitor in the coming two years. Moreover, the company is testing chidamide in NSCLC and breast cancer in early clinical studies.

Chidamide (Epidaza), a class I HDAC inhibitor, was discovered and developed by ChipScreen and approved by the CFDA in December 2014 for the treatment of recurrent of refractory peripheral T-cell lymphoma. Chidamide, also known as CS055 and HBI- 8000, is an orally bioavailable benzamide type inhibitor of HDAC isoenzymes class I 1–3, as well as class IIb 10, with potential antineoplastic activity. It selectively binds to and inhibits HDAC, leading to an increase in acetylation levels of histone protein H3.74

This agent also inhibits the expression of signaling kinases in the PI3K/ Akt and MAPK/Ras pathways and may result in cell cycle arrest and the induction of tumor cell apoptosis.75

Currently, phases I and II clinical trials are underway for the treatment of non-small cell lung cancer and for the treatment of breast cancer, respectively.76 The scalable synthetic approach to chidamide very closely follows the discovery route,77–79 and is described in Scheme 10. The sequence began with the condensation of commercial nicotinaldehyde (52) and malonic acid (53) in a mixture of pyridine and piperidine. Next, activation of acid 54 with N,N0-carbonyldiimidazole (CDI) and subsequent reaction with 4-aminomethyl benzoic acid (55) under basic conditions afforded amide 56 in 82% yield.

Finally, activation of 56 with CDI prior to treatment with 4-fluorobenzene- 1,2-diamine (57) and subsequent treatment with TFA and THF yielded chidamide (VIII) in 38% overall yield from 52. However, no publication reported that mono-N-Boc-protected bis-aniline was used to approach Chidamide.

Photo taken on May 22, 2015 shows a box of Chidamide in Shenzhen, south China’s Guangdong Province. Chidamide is the world’s first oral HDAC inhibitor …

A New Cancer Drug, Made in China

After 14 years, Shenzhen biotech’s medicine is one of the few locally developed from start to finish

Xian-Ping Lu left his research job at a drug maker in the U.S. to co-found a biotech company in his native China.PHOTO: SHENZHEN CHIPSCREEN BIOSCIENCES

HONG KONG— Xian-Ping Lu left his job as director of research at drug maker Galderma R&D in Princeton, N.J., to co-found a biotech company to develop new medicines in his native China.

It took more than 14 years but the bet could be paying off. In February, Shenzhen Chipscreen Biosciences’ first therapy, a medication for a rare type of lymph-node cancer, hit the market in China.

The willingness of veterans like Dr. Lu and others to leave multinational drug companies for Chinese startups reflects a growing optimism in the industry here. The goal, encouraged by the government, is to move the Chinese drug industry beyond generic medicines and drugs based on ones developed in the West.

Chipscreen’s drug, called chidamide, or Epidaza, was developed from start to finish in China. The medicine is the first of its kind approved for sale in China, and just the fourth in a new class globally. Dr. Lu estimates the research cost of chidamide was about $70 million, or about one-tenth what it would have cost to develop in the U.S.

“They are a good example of the potential for innovation in China,” said Angus Cole, director at Monitor Deloitte and pharmaceuticals and biotechnology lead in China.

China’s spending on pharmaceuticals is expected to top $107 billion in 2015, up from $26 billion in 2007, according to Deloitte China. It will become the world’s second-largest drug market, after the U.S., by 2020, according to an analysis published last year in the Journal of Pharmaceutical Policy and Practice.

China has on-the-ground infrastructure labs, a critical mass of leading scientists and interested investors, according to Franck Le Deu, head of consultancy McKinsey & Co.’s pharmaceuticals and medical-products practice in China. “There’re all the elements for the recipe for potential in China,” he said.

But there are obstacles to an industry where companies want big payoffs for a decade or more of work and tremendous costs it takes to develop a drug.

While the protection of intellectual property has improved, China’s cumbersome rules for drug approval and a government effort to cut health-care costs, particularly spending on drugs, could hurt the Chinese drug companies’ efforts, said Mr. Cole of Deloitte.

“Will you start to see success? Of course you will,” said Mr. Cole. However, “I’ve yet to see convincing or compelling evidence that it’s imminent.”

To date, many of the Chinese companies that are flourishing in the life sciences are contract research organizations that help carry out clinical trials, as well as providers of related services.

Some companies, like Shanghai-based Hua Medicine, are buying the rights to develop new compounds in China from multinational drug companies, what some experts consider more akin to an intermediate step to innovation.

Late last year, Hua Medicine completed an early-stage human clinical trial of a diabetes drug in China and in March filed an application to the Food and Drug Administration to develop it in the U.S. as well. The company has raised $45 million in venture funding to date.

Li Chen, who left an 18-year career at Roche Holding AG as head of research and development in China to help start Hua Medicine, said the company’s goal is to “create a game-changer of drug discovery.”

At Chipscreen Biosciences, Dr. Lu and his co-founders set up the company in 2001 in Shenzhen, a city that was quickly growing into a technology and research hub, just over the border from Hong Kong. They created a lab of 10 scientists to use a new analytic technique known as “chemical genomics” to examine the relationships between molecular structures of the existing and failed drugs, how they act on different targets in the body and what genes were being activated or repressed. Now they have more than 60 scientists.

By better predicting how chemicals would act on the body before entering human testing, they hoped they would be more likely get a drug to market.

“How can a small company compete with a multinational?” said Dr. Lu. “The only thing we can compete with is the scientific brain.”

The biggest challenges for the company have been financing and the Chinese regulatory system, said Dr. Lu. The company has raised a total of 300 million yuan ($48 million) over five rounds of venture funding, said Dr. Lu. Chipscreen also receives grant money from the Chinese government.

The company filed its application for approval of chidamide to the Chinese Food and Drug Administration, or CFDA, in early 2013. It had to wait nearly two years for approval, receiving the OK only in December.

Chidamide now is on the market in China for 26,500 yuan ($4,275) a month, a price far lower than patients in the U.S. pay for some of the newest cancer medicines but much more than the typical Chinese patient pays for drugs. Dr. Lu said the price reflects a balance between affordability for patients and return for shareholders. Some investors wanted to price the drug higher.

PAPER

Discovery of an orally active subtype-selective HDAC inhibitor, chidamide, as an epigenetic modulator for cancer treatment

Tumorigenesis is maintained through a complex interplay of multiple cellular biological processes and is regulated to some extent by epigenetic control of gene expression. Targeting one signaling pathway or biological function in cancer treatment often results in compensatory modulation of others, such as off-target drivers of cell survival. As a result, overall survival of cancer patients is still far from satisfactory. Epigenetic-modulating agents can concurrently target multiple aberrant or compensatory signaling pathways found in cancer cells. However, existing epigenetic-modulating agents in cancer treatment have not yet fully translated into survival benefits beyond hematological tumors. In this article, we present a historical rationale for use of chidamide (CS055/Epidaza), an orally active and subtype-selective histone deacetylase (HDAC) inhibitor of the benzamide chemical class. This compound was discovered and successfully developed as mono-therapy for relapsed and refractory peripheral T cell lymphoma (PTCL) in China. We discuss the evidence supporting chidamide as a durable epigenetic modulator that allows cellular reprogramming with little cytotoxicity in cancer treatments.

CLIPS

Chinese scientists develop world’s 1st oral HDAC inhibitor

Lu Xianping works in a lab at Shenzhen Chipscreen Biosciences Ltd. in Shenzhen, south China’s Guangdong Province, May 20, 2015. Lu Xianping, together with other four returned overseas scientists, spent 14 years to develop Chidamide, the world’s first oral HDAC inhibitor, which was given regulatory approval in January. (Xinhua/Mao Siqian)

SHENZHEN, China, Oct. 10, 2013 /PRNewswire/ — GNT Biotech and Medicals Corporation announces the grant of an exclusive license from Shenzhen Chipscreen Biosciences Ltd.for the development and commercialization of Chidamide in Taiwan. Chidamide, an oral, selective histone deacetylase (HDAC) inhibitor, is currently being evaluated in Phase II trials by Chipscreen Biosciences in Peripheral T-Cell Lymphoma (PTCL), Cutaneous T-Cell Lymphoma (CTCL) and Non-Small Cell Lung Cancer patients (NSCLC). GNTbm will develop and commercialize Chidamide primarily in PTCL, NSCLC and will also retain the rights to develop and commercialize Chidamide in other oncology indications in Taiwan.

About Chidamide

Chidamide is a selective HDAC inhibitor against subtype 1, 2, 3 and 10, and being studied in multiple clinical trials as a single agent or in combination with chemotherapeutic agents for the treatment of various hematological and solid cancers. Its anticancer effects are thought to be mediated through epigenetic modulation via multiple mechanisms of action, including the inhibition of cell proliferation and induction of apoptosis in blood derived cells, inhibition of epithelial to mesenchymal transition (EMT, a process that is highly relevant to tumor cell metastasis and drug resistance), induction of tumor specific antigen and antigen-specific T cell cytotoxicity, enhancement of NK cell anti-tumor activity, induction of cancer stem cell differentiation, and resensitization of tumor cells that have become resistant to anticancer agents such as platinums, taxanes and topoisomerase II inhibitors. Chidamide has demonstrated clinical efficacy in pivotal phase II trials on Cutaneous T-Cell Lymphoma (CTCL) and Peripheral T-Cell Lymphoma (PTCL) conducted in China, and is currently undergoing phase II trial in NSCLC together with first line PC therapeutic treatment. Due to its superior pharmacokinetic properties and selectivity, Chidamide may offer better clinical profile over the other HDAC inhibitors currently under development or being marketed.

About GNTbm

GNTbm is a subsidiary of GNT Inc, a Taiwanese company focused on the manufacture of nano-scale metallic particles for food and medical purposes. Founded in 1992 by a team of electronic professionals, GNT has successfully developed the innovative technology of physical metal miniaturization based on the patent of MBE (Molecular Beam Epitaxy). Further information about GNT Inc is available at www.gnt.com.tw.

GNTbm was established in August 2013, and housed in the Nankang Biotech Incubation Center, (NBIC), in Nankang, Taipei. Lead by Dr. Chia-Nan Chenalong with an experienced team of scientists, GNTbm will explore development and commercialization of novel drug delivery systems, Innovative biomedical and diagnostic tools based on gold nanoparticles.

About Shenzhen Chipscreen Biosciences Ltd.

Chipscreen is a leading integrated biotech company in China specialized in discovery and development of novel small molecule pharmaceuticals. The company has utilized its proprietary chemical genomics-based discovery platform to successfully develop a portfolio of clinical and preclinical stage programs in a number of therapeutic areas. Chipscreen’s business strategy is to generate differentiated drug candidates across multiple therapeutic areas. Drug candidates are either developed by Chipscreen or co-developed and commercialized in a partnership at the research, preclinical and clinical stages. The company was established as Sino-foreign joint venture in 2001. Further details about Chipscreen Bioscience is available atwww.chipscreen.com.

Beijing Shenogen Biomedical announced that Icaritin, a China Class I cancer drug, was granted Fast Track Review status after the company filed its New Drug Approval submission to the Beijing Food & Drug Administration. Icaritin is an oral traditional Chinese medicine, derived from barrenwort, which targets the estrogen receptor α36. Shenogen has conducted clinical trials of Icaritin in patients with liver cancer, though it expects the drug will also prove effective in breast cancer and other estrogen-related cancers as well. More details…. http://www.chinabiotoday.com/articles/20150917

Cardiovascular function improvement, hormone regulation and antitumor activity.
2. The anti-MM activity of Icaritin was mainly mediated by inhibiting IL-6/JAK2/STAT3 signaling.
3. The inhibitory activity of Icariside II on pre-osteoclast RAW264.7 growth was synergized by Icaritin, which maybe contribute to the efficiency of Herba Epimedii extract on curing bone-related diseases, such as osteoporosis.
4. The Icaritin at low concentration (4 or 8 μmol/L) can promote rat chondrocyte proliferation and inhibit cell apoptosis, while the effect of Icaritin on rat chondrocyte at high concentration was reversed.
5. Icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.
6. Icaritin dose-dependently inhibits ENKL cell proliferation and induces apoptosis and cell cycle arrest at G2/M phase. Additionally, Icaritin upregulates Bax, downregulates Bcl-2 and pBad, and activates caspase-3 and caspase-9.

What is Epimedium ?

Herba epimedii (Epimedium, also called bishop’s hat, horny goat weed or yin yang huo), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. It is a genus of about 60 flowering herbs, cultivated as a ground cover plant and an aphrodisiac. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Most of them bloom in the early spring, and the leaves of some species change colors in the fall, while other species retain their leaves year round.

Figure 1 Epimedium

Epimedium Raw Material

The herbs we used to extract icariin is one species of Epimedium, which name is Epimedium brevicornum Maxim. This kind of epimedium only can be abundantly found in Gansu province of China. And because of the growth habit of this kind of herb, which only grows under trees, it can’t to be planted, only can harvest the wild one.

This wild epimedium contains quite a bit of active components, depending on its long growth time and rich nutrient. Usually the content of the icariin is not lower than 1%.

Below photo is the herb specimen which we use. Picking in the epimedium full-bloom stage. And the medicinal value of the herb is the best at this time. The herb we select contains roots, stems, leaves and flowers. And we extract with the whole herb.

Figure 2 Epimedium for extract

Epimedium Extract

Epimedium extract is a herbal supplement claimed to be beneficial for the treatment of sexual problems such as impotence. It is believed to contain a number of active components, including plant compounds that may have antioxidant activity and estrogen-like compounds. The major components of Epimedium brevicornum are icariin, epimedium B and epimedium C. It is reported to have anti-inflammatory, anti-proliferative, and anti-tumor effects. It is also reported to have potential effects on the management of erectile dysfunction.

Figure 3 HPLC spectrum of icariin

Our specification available is Icariin HPLC 50%- 98%. Below please see the the information for reference:

Figure 4 Epimedium Extract(Icariin)

Derivatives

The plant extracts of epimedium traditionally used for male impotence, and the individual compounds is icariin, were screened against phosphodiesterase-5A1 (PDE5A1) activity. Human recombinant PDE5A1 was used as the enzyme source. The E. brevicornum extract and its active principle icariin were active. To improve its inhibitory activity, some derivatives ware subjected to various structural modifications, which include icaritin, icariside II and 3,7-bis(2-hydroxyethyl) icaritin. There have some scientific papers report that the improved pharmacodynamic profile and lack of cytotoxicity on human fibroblasts make such compounds a promising candidate for further development. We hope that our new products can help you to find more commercial opportunity.

In this way, we can introduce those products as below, and we can also provide more details about the products according to your demand. The 1H-NMR of icaritin and 3,7-bis(2-hydroxyethyl) icaritin is as below.

Epimedium has been used to treat male erectile dysfunction in Traditional Chinese Medicine for many centuries. The main functions of Epimedium brevicornum in ancient Chinese books focused on the nourishment of kidney viscera and reinforcement of ‘yang’, resulting in the restoration of erectile function in males.

The novel total synthesis of icaritin (1), naturally occurring with important bioactive 8-prenylflavonoid, was performed via a reaction sequence of 8 steps including Baker-Venkataraman reaction, chemoselective benzyl or methoxymethyl protection, dimethyldioxirane (DMDO) oxidation, O-prenylation, Claisen rearrangement and deprotection, starting from 2,4,6-trihydroxyacetophenone and 4-hydroxybenzoic acid in overall yields of 23%. The key step was Claisen rearrangement under microwave irradiation. MS, 1H and 13C NMR techniques have been used to confirm the structures of all synthetic compounds. – See more at: http://www.eurekaselect.com/124334/article

The present invention relates to compositions comprising icariside I, and to a novel, one step method of preparing such compositions, comprising converting specific prenylated flavonol glycosides such as epimedium A, epimedium B, epimedium C, icariin, and their corresponding acetate derivatives contained in an Epimedium plant extract to a single compound, namely icariside I shown below as compound I, which was surprisingly discovered to be a strong PDE-5 inhibitor.

This invention further comprises compositions enriched for anhydroicaritin, and to methods of preparing such compositions. One method of this invention for preparing compositions enriched for anhydroicaritin comprises a one-step method of converting prenylated flavonol glycosides, specifically the sagittatoside compounds A, B, and C, and the corresponding acetate derivatives, present in Epimedium plant extracts to a single compound, namely anhydroicaritin shown below as compound II, which was also discovered to be a strong PDE-5 inhibitor.

EXAMPLES Example 1 Acid Hydrolysis of a 50% EtOH Extract and Purification by Reversed Phase ChromatographyWhole Epimedium grandiflorum leaves were extracted with a 1:1 mixture of ethanol and water at 55° C. The resulting extract (referred to as a “50% EtOH extract”) was filtered and the filtrate concentrated at 40-50° C. under vacuum and then dried under vacuum at 60° C. to a dry solid. The dried extract (131 g) containing approximately 5.8 g of total PFG’s was placed in a 2 liter round bottom flask and 1 L of 90% ethanol was added. The mixture was heated to reflux to help dissolve the solids. Concentrated sulfuric acid (28 mL) was added. The mixture refluxed for 2 hr, cooled to room temperature, and 900 mL of water added with stirring. Next the mixture was filtered using vacuum to remove insoluble sulfate salts and other solids and loaded on a 2.5×56 cm (275 mL) column packed with 250-600 micron divinylbenzene cross-linked polystyrene resin (Mitsubishi Chemical). The column was washed with 2 column volumes (CVs) of 60% ethanol and the icariside I was eluted with 2 CVs of 95% ethanol. The product pool was air-dried producing 11.3 g of brown solids. HPLC analysis (FIG. 5) showed that the solids contained 18% icariside I (peak 15.27 min) and 12% anhydroicaritin (peak 25.15 min). The recovery of the icariside I in the product pool was 87% of the amount present in the hydrolyzate.

Example 2 Purification of a Hydrolyzate by Liquid/liquid ExtractionThe ethanolic hydrolyzate (25 mL) prepared in Example 1 was mixed with 62.5 mL of de-ionized water and the pH was adjusted to 7.0 using 50% (w/w) sodium hydroxide solution. The resulting mixture was extracted with three 25 mL portions of ethyl acetate and the combined ethyl acetate extracts were back extracted with 150 mL of water. The ethyl acetate layers were combined, dried, and assayed for icariside I. HPLC analysis (FIG. 6) showed that the dried EtOAc fractions contained 22% icariside I (peak 15.29 min) and 11% anhydroicaritin (peak 25.27 min), and icariside I recovery into the ethyl acetate was 97% of the amount present in the hydrolyzate. The partition coefficient for icariside I between ethyl acetate and water was found to be 16, indicating that the icariside I has a high affinity for ethyl acetate over water.

Example 3 Acid Hydrolysis of a 50% EtOH Extract and Purification by PrecipitationThe dried extract (204 g) described in Example 1 was mixed with 1 L of 90% EtOH and then heated to reflux to help dissolve the solids. Sulfuric acid (25 ML) was added slowly with swirling. The mixture was refluxed 90 minutes and immediately chilled to stop the reaction. After cooling to room temperature, the mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials, and the cake was washed with about 350 mL of 90% ethanol. The resulting ethanolic hydrolyzate (1.34 L) contained 4.1 g of icariside I.

The ethanolic hydrolyzate prepared above (1.32 L) was placed in a 10 L container and 40 g of 50% (w/w) sodium hydroxide solution was added followed by 20 mL of phosphoric acid. Next 3.3 L of deionized water was added with stirring. The pH of this mixture was 2.4. Sodium hydroxide solution (50% w/w ) was added until the pH was 8.25. The mixture was heated to 65° C. to assist with the coagulation of the precipitate. The mixture was cooled to room temperature and stirred for 0.5 hr at room temperature before filtering through a cellulose filter using vacuum. The resulting brown solids were washed with 715 mL of 10% ethanol and dried either under vacuum at room temperature or in air at 55° C. to yield brown solids. HPLC analysis (FIG. 7) showed the solids contained 20% icariside I (peak 15.27 min) and 10% anhydroicaritin. Recovery of icariside I using this precipitation procedure was 94% of the amount present in the hydrolyzate.

Example 4 Acid Hydrolysis of a Water Extract and Purification by PrecipitationGround Epimedium grandiflorum leaves (0.40 kg) were mixed with 5 L water in a 10 L round bottom flask. The flask was placed on a rotary evaporator for two hours at a rotation speed of 120 rpm and a water bath temperature of 90° C. The extract was filtered under reduced pressure through cellulose paper. The resulting filtrate (3.2 L) was evaporated using the rotary evaporator to a volume of 100 mL and dried under vacuum at 50° C.

The dark brown solids prepared above (40.4 g) were mixed with 200 mL of 90% ethanol and 6.0 mL of sulfuric acid in a 500 mL round bottom flask. The mixture was refluxed for 90 minutes and immediately chilled to stop the reaction. This mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with 15 mL of 90% ethanol. The resulting ethanolic hydrolyzate (215 mL) contained 0.53 g of icariside I.

The hydrolyzate prepared above (50 mL) was transferred to a 250 mL beaker and 2.5 mL of 50% (w/w) sodium hydroxide solution was added with stirring to adjust the pH of the solution to pH 9, followed by 1.5 mL of concentrated phosphoric acid. Deionized water (125 mL) was added, and the mixture was adjusted to pH 8.2 using 1.5 mL of 50% sodium hydroxide solution. The mixture was heated to 65° C. to assist with coagulation of the precipitate and cooled to room temperature. The mixture was allowed to sit undisturbed at room temperature for 30 minutes prior to filtration under reduced pressure through cellulose paper. The resulting olive-green solids were washed with 25 mL of de-ionized water and dried under vacuum at room temperature or in air at 80° C. to produce olive-green solids. HPLC analysis (FIG. 8) showed the solids contained 60% icariside I (peak 15.33 min) and 2.4% anhydroicaritin (peak 25.40 min). Recovery of icariside I using this precipitation procedure was 92% of the amount present in the hydrolyzate.

Example 5 Enzymatic Hydrolysis of Icariside Ia) The substrate was a partially purified icariside I product with 20% icariside I and 11% anhydroicaritin. About 50 mg was dissolved in 10 mL of ethanol, and water or buffer was added until the mixture became cloudy (about 20% ethanol). The following dry enzymes were added to separate samples: α-amylase, α-glucosidase, β-amylase, β-glucosidase, hesperidinase, lactase, and pectinase. The samples were incubated overnight at 40 ° C. and analyzed by HPLC. The results were only semi-quantitative due to the difficulty in dissolving the anhydroicaritin that precipitated from the samples. However, several of the chromatograms did show a definite reduction in icariside I and increase in the ratio of anhydroicaritin to icariside I. The best results were obtained using hesperidinase, lactase, β-glucosidase and pectinase.

A larger scale experiment was done using hesperidinase in order to isolate pure anhydroicaritin for characterization. Pure icariside I (20 mg )was dissolved in 10 mL of ethanol and 50 mL of water and 200 mg of hesperidinase enzyme was added and the mixture was incubated for 24 hr at 40 ° C. Crude anhydroicaritin was collected via filtration and purified on a 2.5×30 cm semi-prep C-18 HPLC column using a gradient of 50:50 (MeCN/H2O) to 80:20 (MeCN/H2O) in 20 min. The pure anhydroicaritin was analyzed by LC/MS and proton NMR.

b) Enzymatic Hydrolysis of PFG’s: The purified PFG solids (55.3%, purified by reversed-phase chromatography of a 50% EtOH extract) were subjected to enzymatic hydrolysis with the same enzymes and conditions described in part (a). Hesperidinase, lactase, β-glucosidase and pectinase appeared to convert the mixture of PFG’s to a mixture of sagittatosides, but no icariside I or anhydroicaritin were observed. This indicated that these enzymes were specific for the 7-β-glucosyl group and did not hydrolyze the 3-position sugar(s).

Example 6 Preparation of a High Anhydroicaritin-containing ProductA high sagittatosides Epimedium sagittatum extract containing 24.7% total sagittatosides (assayed as icariin) and 8.1% icariin and other expected prenylated flavonol glycosides was obtained from China. A 50 g portion of this extract was mixed with 250 mL of 90% ethanol and 7.5 mL of concentrated sulfuric acid in a 500 mL round bottom flask. The mixture was refluxed for 90 minutes, then allowed to cool to room temperature. The hydrolyzed mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with approximately 20 mL of 90% ethanol. The resulting filtered ethanolic hydrolyzate (305 mL) contained 3.75 g of anhydroicaritin and 2.50 g of icariside I.

The filtered hydrolyzate prepared above (200 mL) was transferred to a 1000 mL container and 8.0 mL of 50% (w/w) sodium hydroxide solution was added with stirring, followed by 4.0 mL of phosphoric acid. De-ionized water (500 mL) was then added. This mixture was adjusted to pH 4.9 using 50% sodium hydroxide solution. The mixture was allowed to sit undisturbed at room temperature for 24 hours prior to decanting off the liquid. The resulting solids were macerated using de-ionized water and filtered under reduced pressure through cellulose paper. The resulting dark brown solids (11.9 g) were washed with de-ionized water and dried in air overnight. The dark brown solids contained 20% anhydroicaritin and 12% icariside I and an anhydroicaritin/icariside I ratio of 1.66. The recovery of anhydroicaritin in the precipitation procedure was 94% from the hydrolyzate.

Example 7 Recrystallization of Icariside IIcariside 1 (30 mg) obtained by a method described in Example 1 was dissolved in a minimum of hot tetrahydrofuran (THF). Hot methanol (approximately 10 mL) was then added. The hot THF/MeOH solution was filtered through a PTFE filter into a vial and allowed to evaporate at room temperature to about 5 mL, whereupon crystals began to form, and then placed in a 4° C. refrigerator for 24 hours. The crystals were filtered and washed with cold methanol and dried in a vacuum. Icariside I (21 mg) was isolated as yellow crystals and had a chromatographic purity of 97.4%.

Example 8 Large Scale Acid Hydrolysis of an Epimedium extractAn 800 g portion of an Epimedium sagittatum powder extract obtained from China containing about 13% total prenylflavonol glycosides as icariin was mixed with 4.0 L of 90% ethanol and 120 mL of sulfuric acid in a 10 L round bottom flask. The mixture was refluxed for 90 minutes and immediately chilled to stop the reaction. This mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with approximately 200 mL of 90% ethanol. The resulting ethanolic hydrolyzate (4.0 L) contained 33.7 g of icariside I.

The ethanolic hydrolyzate prepared above was transferred to a 34 L container and 200 mL of 50% (w/w) sodium hydroxide solution was added with stirring, followed by 120 mL of phosphoric acid. De-ionized water (10 L) was then added. This mixture was adjusted to pH 8.2 using 120 mL of 50% sodium hydroxide solution. The mixture was stirred for 10 minutes and allowed to sit undisturbed at room temperature for 60 minutes prior to filtration under reduced pressure through cellulose paper. The resulting olive-green solids were washed with 750 mL of de-ionized water and dried under vacuum at 50° C. or in air at 80° C. The olive-green solids contained 44.6% icariside I. Recovery of icariside I in the precipitation procedure was 96% from the hydrolyzate.

Example 9 Large Scale Purification of an Epimedium Extract Containing Prenylflavonoid GlycosidesA 3.7 kg portion of an Epimedium sagittatum powdered extract obtained from China containing approximately 10% total prenylflavonol glycosides (PFG’s) assayed as icariin was stirred with 35 L of 85/15 acetone/water (v/v) in a 50 L mixing tank. The mixture was stirred vigorously for 30 minutes and allowed to sit for 5 minutes. The acetone extract layer (36 L) was decanted from the tank and contained 362 g of PFG’s. Recovery of the PFG’s in this extraction procedure was 96%.

A portion (about 500 mL) of the acetone extract was dried under reduced pressure at 50° C. or less, providing 16.1 g of brown solids which were analyzed to contain 28.6% total PFG’s when assayed as icariin.